The long-term objective of this proposal is to establish a unified theory to describe the mechanisms of crosstalk signaling between Ca2+ and reactive oxygen species (ROS) in cardiac muscle cells, and to translate these signaling mechanisms to the physiology and pathology of cardiac function. The pivotal role of mitochondrial Ca2+ and ROS in mediating the life and death of cardiac muscle cells is well recognized. The separation between mitochondria-mediated life versus death resides in the finest balance between concentrations of Ca2+ and ROS. The majority of existing research in the field focuses individually either on Ca2+ or ROS homeostasis. The interaction between these two signaling pathways, however, has just begun to gain attention by a small number of laboratories. Intriguingly, there is a growing library of literature suggesting that mitochondrial dynamics (fission, fusion, and trafficking) play an essential role in the physiological regulation of cellular ATP, Ca2+, and ROS homeostasis. In this proposal, we will study how these three important components (Ca2+, ROS, and mitochondrial fission machinery) communicate to regulate cardiac Ca2+ and ROS crosstalk signaling. We will use a multidisciplinary approach encompassing techniques of cell biology (e.g. confocal microscopy), molecular biology (e.g. gene transfer), biochemistry (e.g. western blots), and transgenic mouse models (e.g. cyclophilin D (CypD) knockout mice and mt-cpYFP transgenic mice) to elucidate the mechanisms of ROS and Ca2+ symbiosis with an unique emphasis on mitochondrial fission protein DLP1 and mitochondrial permeability transition (MPT). Our central hypothesis is: an increased mitochondrial Ca2+ concentration ([Ca2+]m) favors the balance of mitochondrial dynamics towards fission that in turn increases ROS generation. The resulting oxidized environment leads to additional mitochondrial Ca2+ influx. Both the increases in [Ca2+]m and ROS enhance the opening probability of MPT that further augments ROS generation. Eventually, this positive feedback loop is counter balanced by Ca2+ and ROS activated mitochondrial Ca2+ efflux mechanisms including Na/Ca exchange and MPT. The three specific aims are: 1) To determine whether an increased [Ca2+]m promotes mitochondrial fission processes, which then lead to increase ROS generation. 2) To assess the contribution of a CypD containing MPT pathway in [Ca2+]m-mediated ROS generation. 3) To determine the role of MPT as a rapid Ca2+ efflux mechanism of mitochondria.